The monitoring of bovine pregnancies derived

Reprod. Nutr. Dev. 42 (2002) 613–624
© INRA, EDP Sciences, 2003
DOI: 10.1051/rnd:2002047
The monitoring of bovine pregnancies derived
from transfer of in vitro produced embryos
Marcel A.M. TAVERNEa*, Simone P. BREUKELMANa, Zsolt PERÉNYIb,
Steph J. DIELEMANa, Peter L.A.M. VOSa, Herman H. JONKERa,
Jean-François BECKERSb
Department of Farm Animal Health, Faculty of Veterinary Medicine,
Utrecht University, The Netherlands
b Department of Physiology of Reproduction, Faculty of Veterinary Medicine,
University of Liège, Belgium
c Holland Genetics, R&D, Arnhem, The Netherlands
Abstract — Both an increased rate of embryonic, foetal and perinatal losses, and the occurrence of
deviations in foetal and placental development are associated with bovine pregnancies obtained
from in vitro produced embryos. This thus requires for a more accurate and frequent monitoring of
foetal and maternal functions during pregnancies. Such approaches will enable to establish the period
during which these losses and deviations in development occur and to plan possible clinical interventions. This paper reviews some recent data on return rates, late embryonic and foetal losses in recipients after the transfer of either MOET, IVF or nuclear transfer embryos. Special attention is paid to
the diagnostic value of measurements of pregnancy specific/associated proteins and progesterone in
maternal plasma. Possibilities to measure foetal body sizes, size of placentomes and foetal heart rate
by means of transrectal or transabdominal ultrasonography are illustrated with data from the literature and with recent results from our own large field study with MOET, IVP-co-culture and IVP-SOF
cow / pregnancy / pregnancy proteins / ultrasonography / foetus / placenta / foetal heart rate /
embryo transfer / in vitro fertilization / nuclear transfer
* Correspondence and reprints
E-mail: [email protected]
M.A.M. Taverne et al.
In cattle breeding, the interest of collecting data on the course of a pregnancy in
individual females generally subsides, once
a positive pregnancy diagnosis has been
made within two months after mating or
artificial insemination. The same is true for
the pregnancies obtained after embryo transfer, despite the fact that considerable efforts
and costs have sometimes to be made to
reach a pregnancy in the first place. Attention to pregnant females is only resumed at
the time of calving, when it needs to be
judged if delivery can take place spontaneously or should be accomplished by artificial means to improve the chances for survival of the calf. Observations and data
connecting (pathophysiological) events during (early) pregnancy and parturition with
the morbidity and performance of calves at
later life are not routinely available. However, such an approach appears to be more
relevant keeping in mind the recently pronounced concept [1–3, 45] that several diseases encountered during adult life might
have their origin during the period of foetal
development. The same might be true for
physical performance and (re)production in
domestic species [37, 39, 40, 48]. In this
respect one seems to have forgotten that,
within the context of research on growth
and production of farm animals, Grahm
Everitt already warned in 1968 that “The
extent to which events of later life may be
modified by factors operating during the
formative stages appears insufficiently
appreciated”(see Bell [4]). The so-called
“Large Calf Syndrome” [7, 24, 64] might
be an undesirable example of such types of
long-term effects.
Early reports on increased embryonic and
foetal losses and the variable occurrence of
deviant foetal development during pregnancies obtained from in vitro produced
embryos (reviewed by Kruip en den Daas
[35]), have stimulated the interest for
research on the prenatal development of farm
animal species, especially in ruminants.
Besides many (ongoing) studies to unravel
the cellular and molecular mechanisms
of deviant development of IVF-derived and
cloned embryos [11, 41, 53, 64], several
authors have reported on placental and foetal
growth and pathophysiological events during the perinatal period [18, 19, 22, 26, 29,
30, 47, 49, 51, 59, 60]. The bovine foetus
and placenta are not easily accessible for
(on farm) investigations, especially during
the second and third trimester of gestation.
Yet, there is a need for more intensive monitoring of pregnancies, not only to collect
more accurate information on the exact timing of prenatal losses, but also to document
or predict deviations in prenatal development. Such approaches will contribute to
the understanding of the mechanism of disturbed development and will allow timely
clinical interventions, either during pregnancy or at parturition. This contribution
will therefore concentrate on late embryonic, foetal and perinatal losses resulting
from pregnancies derived from transfers of
IVF-derived and cloned embryos, with a
special focus on methods by which such
pregnancies can be more intensively monitored. Our own data from a field study of
our own group, in which we compared the
course and outcome of IVP and MOET
pregnancies, will be presented as well.
2.1. Pregnancies after transfer
of IVP-embryos
When recipients show a regular return to
oestrus (between 19 and 22 days after their
first oestrus), it can be assumed that early
embryonic mortality occurred, i.e. before
maternal recognition of pregnancy took
place. Both after A.I. and embryo transfer of
in vivo or in vitro produced embryos, high
rates of such early embryonic losses have
been reported [17, 25, 38, 46, 54], especially
when quality grade 2 embryos had been used
Monitoring of bovine ET-pregnancies
for transfer. Recently, Heyman et al. [26]
concluded on the basis of plasma progesterone levels, that on day 21, no significant
differences in the percentages of presumed
pregnancies existed between groups of recipients to which either cloned or IVF embryos
had been transplanted.
Immediately after this period during
which regular returns can be expected, the
presence of a pregnancy can be presumed
(on the basis of plasma progesterone profiles or irregular returns) or diagnosed for
the first time (by means of ultrasonography
or measurements of plasma pregnancy proteins). When a positive early pregnancy
diagnosis is followed by a return to oestrus
between days 23 and 42, late embryonic
mortality has occurred. Under field conditions, however, the first pregnancy diagnosis usually takes place only during the foetal
period (around day 40). This means that it is
usually very difficult to derive accurate figures on early and late embryonic mortality
from published data on pregnancy rates. In
their world-wide retrospective review on
the results from transfers of IVP embryos,
Kruip and den Daas [35] reported that while
70% of the embryonic losses after A.I.
or ET occurred within the first 21 days of
fertilization, this was only 58% for IVP
embryos. This might be associated with a
more strict selection of good quality IVP
blastocysts. A more detailed picture can
be obtained from the data published by
van Wagtendonk et al. (2000). These authors
compared return rates and pregnancy
outcome after (single) transfers of either a
MOET, IVP-co-culture or IVP-SOF embryo
(Tab. I).
Total return rates were not significantly
different between the three groups, but
the return rate between 0 and 31 days was
higher in the recipients with a MOET or
IVP-SOF embryo. However, the relative
proportion of returns between days 24 and
31 was significantly higher in the IVP-coculture group, while losses beyond day 52
were also higher in this group (although not
significantly different). In a recent paper
[36] pregnancy losses occurring between
days 38 and 90 were found to be 11.2% and
9.9% for lactating dairy cows, inseminated
after spontaneous or synchronized oestrus
In a recent field study, we monitored
groups of recipients with similar types of
embryos (for details of production: see
van Wagtendonk-de Leeuw et al. [60]) by
regular blood sampling between day 7 and
day 119 after embryo transfer. Around day
35, transrectal ultrasonography took place
for pregnancy diagnosis and in 65 of these
recipients ultrasonography was repeated at
weekly intervals for foetometry and measurements of foetal heart rate (see below).
The data are presented in Table II.
Failure of pregnancy without an observed
return to oestrus occurred in 3.2%, 4.4% and
4.5% of the recipients of the MOET, IVP
co-culture and IVP-SOF group, respectively.
Although the percentage of pregnancy
failures was slightly higher in the IVP-SOF
Table I. Returns to oestrus after transfer of a single MOET, IVP-co-culture or IVP-SOF embryo to
recipients of the herd of Holland Genetics (after van Wagtendonk-de Leeuw et al. [60]).
(n = 465)
Total return (%)
Returns between days 0–31 as % of total returns
Returns between days 24–31 as % of day 0–31 returns
Returns between days 32–52 as % of total returns
Returns beyond day 52 as % of total returns
IVP-co-culture IVP-SOF
(n = 157)
(n = 101)
M.A.M. Taverne et al.
Table II. Early and late returns to oestrus of recipients to which either a MOET, IVP-co-culture or
IVP-SOF embryo had been transferred (Perényi et al., in preparation).
(n = 118)
Total % pregnancy failures
Returns before day 24 as a % of total pregnancy failures
Returns between days 24 and 119 as a % of total
pregnancy failures
Returns beyond day 119 as a % of total pregnancy failures
group and no late foetal losses occurred in
these recipients, there were no differences
between the three groups as to the relative
proportions of early embryonic losses
(before day 24) and late embryonic plus
early foetal losses (between days 24 and
119). Overall, about one third of all transfers
failed before day 24, while slightly more
than one fifth of the transfers resulted in
losses between days 24 and 119.
On the basis of plasma progesterone (P4:
see Dieleman and Bevers [15]) and PAG1
(see Zoli et al. [65]) profiles of these animals, we were able to analyse the pregnancy
failures between day 24 and 119 in more
detail. By using plasma levels from ongoing
pregnancies with calvings at term as reference values, decreases of P4 and PAG1 levels below one tailed 95% confidence intervals resulted in the attribution of each case
of pregnancy failure with a complete data
file (n = 56) to one of the following three
A: recipients in which a drop or subnormal concentration occurred earlier in the P4
than in the PAG1 profile.
B: recipients in which a drop or subnormal concentration occurred earlier in the
PAG than in the P4 profile.
C: recipients in which subnormal hormone concentrations or decreases occurred
at (about) the same time.
Typical examples of plasma profiles from
recipients of group A and B are presented in
Figures 1A and 1B.
IVP-co-culture IVP-SOF
(n = 44)
(n = 106)
Table III presents the distribution of these
three types of pregnancy failures, occurring
between day 24 and 119, over the three
groups of recipients.
These data indicate that (a) type A was
the dominant type of failure in each group of
recipients, and (b) type B losses occurred
slightly more frequent in recipients with
IVP-SOF embryos. If one accepts that
PAG1 profiles reflect the viability of the
trophoblast/placenta [55, 56], this would
implicate that in pregnancies which have
already passed the stage of maternal recognition of pregnancy, failures after transfer
of IVP-SOF embryos are more often the
result of disturbed conceptus development.
In this respect it is interesting to note that
impaired vascularization of the allantois has
been more often associated with conceptuses derived from in vitro produced ruminant embryos [14, 28, 44].
We were not able to detect significant
differences in plasma P4 and PAG1 levels
between the three groups of recipients during the first 4 months of ongoing pregnancies [62], but we did not find differences
between the mean birth weights of calves
from these three groups. Yet, an increased
foetal weight [17] and increased placental
weight [5, 17] have already been reported
during midgestation in the recipients of
IVP embryos. It appears that the dramatically increased plasma levels of PAG during
the final weeks of gestation are better
correlated with foetal weight [51] than the
Monitoring of bovine ET-pregnancies
levels during early pregnancy. However, it
remains to be investigated to what extent
the reduced number of placentomes, with a
much larger individual size, (which have
been found in IVP pregnancies by Bertolini
and Anderson [5, 6]), do affect plasma PAG
levels. Compared to placentas from foetuses
derived from in vivo embryos, the relative
Figure 1. Plasma progesterone (P4) and PAG1 levels in two cases with an early pregnancy loss.
(A) a recipient, pregnant after transfer of a MOET embryo but returning to oestrus on day 42 after the
reference oestrus; a decline of plasma P4 precedes a decrease in PAG1 levels; (B) a recipient, pregnant after transfer of a IVP-SOF embryo but returning to oestrus on day 49 after reference oestrus;
a decline in the PAG1 level precedes the drop in P4 [43].
Table III. Distribution of three types of pregnancy losses among the three categories of recipients which
returned to oestrus between days 24 and 119.
Type A pregnancy loss (n = 36)
Type B pregnancy loss (n = 16)
Type C pregnancy loss (n = 4)
(n = 22)
(n = 7)
(n = 25)
17 (77%)
5 (23%)
5 (71%)
1 (14.5%)
1 (14.5%)
14 (56%)
18 (32%)
13 (12%)
M.A.M. Taverne et al.
abundance of binucleate cells, by which the
pregnancy proteins are produced, have been
found in the placentas from in vitro embryos
on day 63 [20], although the same authors
previously reported a lower volume density
of binucleate cells in the placentas of IVP
foetuses at day 222 [18]. There is clearly a
need for more detailed studies on the possible relationships between gross morphology (number and size of placentomes), vascularization, cellular differentiation and
production of proteins and hormones of the
bovine placenta during normal A.I., MOET
and IVP pregnancies (see also below).
It has been described [8, 12] that transrectal ultrasonography can be used to visualize the details of the bovine conceptus at
very early stages (before day 24). However,
reliable quantification of ultrasonographic
details of the development of the conceptus is only possible at a later stage [6, 13,
23, 33]. Especially the measurement of
foetal body structures (CRL: crown rump
length; BPD: biparietal diameter of the cranium; CAU: cross section of the abdomen)
can be expected to reflect both the retardation or enhancement of foetal growth.
Recently, Bertolini and coworkers [6]
reported that the CRL of foetuses, derived
from IVP-co-culture beef cattle embryos,
had a significantly reduced CRL between
days 37–58 when compared to foetuses from
MOET embryos, while at birth the mean
body weight of the IVP calves (n = 6) was
significantly higher.
Because it has frequently been reported
that pregnancies derived from IVP embryos
suffer from a higher incidence of both prenatal losses and result more often in heavy
foetuses and offspring, we also performed
repeated ultrasonographic measurements
of foetuses. Between days 35 and 119 foetal
growth was compared between pregnancies
resulting from the transfer of MOET,
IVP-coculture and IVP-SOF embryos.
Reports from early pregnancies in women
suggest that deviations from normal foetal
heart rate (FHR) may be predictive for future
foetal losses or for foetuses with chromosomal abnormalities [50, 57]. Reports on
abnormal vascularization of the allantois
during early pregnancy [14, 44] and data on
deviant cardiac structure and function in
some foetal and newborn ruminants resulting from IVP embryos [27, 52, 60] require
more studies on prenatal cardiovascular
function. This is supported by the recent
finding of Bertolini et al. [6] that FHR of
IVP-concepti (n = 6) was significantly
higher between days 37 and 93 of gestation
when compared with MOET (n = 6) foetuses. We therefore also included measurements of FHR in our study with three groups
of bovine pregnancies [9, 10]. At weekly
intervals, transrectal scans were video-taped
and measurements of BPD, CAU (at the
insertion of the umbilical cord) and CRL
and calculations of FHR were performed
afterwards. It appeared that repeated and
reliable measurements of CRL of the same
foetus were less often possible during transrectal scanning than with the other two
types of foetometry. In addition, the measurement of the CRL appeared less reliable
in our hands, because bending of the foetal
spine by foetal movements appeared to
cause a considerable variation of the CRL
during a single scanning session. FHR data
are presented in Figure 2.
We found no significant differences in
BPD, CAU and FHR between the three
groups of foetuses between days 35–110 of
pregnancy. However, the mean birth weights
of calves resulting from these three different
types of embryos were not significantly different either. Also when BPD, CAU and
FHR were retrospectively compared between
calves with a birth weight below 40 kg and
those weighing more than 51 kg, no significant differences in these foetal parameters
were found.
Changes in FHR followed a parabolic
curve, different from the initial data published by Curran et al. [13], but comparable to the curve that was based on a limited
group of A.I. foetuses by Ginther [23]. Plots
Monitoring of bovine ET-pregnancies
FHR (bpm)
gestational age (days)
Figure 2. Plots of individual (open symbols) calculated Fetal Heart Rates and LS-means (closed
symbols and with lines of best fit) for three groups of foetuses; circles: MOET foetuses (n = 25);
squares: IVP-co-culture foetuses (n = 14); rhombs: IVP-SOF foetuses (n = 22) [9].
of trend lines for FHR of our individual foetuses demonstrated that in some of the pregnancies that did not proceed until term, FHR
was outside the 95% confidence intervals.
This indicates that it might be useful to further explore FHR, based on more frequently
performed measurements, as an early indicator of future foetal death in cattle.
2.2. Pregnancies after transfer
of cloned embryos
Because embryonic and foetal losses are
considerably higher and abnormal foetal
development and increased birth weight
occur more often in pregnancies after the
transfer of cloned embryos [35, 47, 63], it
appears even more appropriate to use intensive ultrasonographic and biochemical monitoring in recipients of cloned embryos.
Ectors et al. [16] demonstrated that PAG
levels in heifers pregnant from transferred
nuclear transfer embryos were higher during
late pregnancy than in recipients of IVF
embryos. In a recent paper from France [26]
monitoring took place during pregnancies
obtained after transfer of cloned (somatic
adult, somatic foetal or embryonic) and
IVF-co-culture embryos. As already indicated above, early embryonic losses did not
appear to be increased after the transfer of
cloned embryos, as judged by plasma progesterone levels on day 21. However, at the
time of the first and the second transrectal
ultrasound scan, significantly less recipients with somatic clones appeared pregnant.
Data on the percentage of pregnant animals
at different stages of the first trimester and
the final calving rates are summarized in
Table IV.
Foetal survival decreased dramatically,
especially in the recipients who received an
embryo cloned from adult somatic cells.
Foetal deaths were not observed beyond
day 90 in recipients of the control IVF or
embryonic cloned groups.
M.A.M. Taverne et al.
Table IV. Data on pregnancies of recipients to which either a cloned embryo (3 different types) or an
IVF-co-culture embryo had been transferred (after Heymann et al. [26]).
% presumed pregnant D21
% found pregnant D35
% found pregnant D50
% found pregnant D70
% found pregnant D90
% calves at term
cloned from
somatic adult
cells (n = 133)
cloned from
somatic foetal
cells (n = 40)
cloned from
cells (n = 67)
(n = 51)
* Including one twin from a single embryo transfer.
Similar to our own findings on PAG1
levels during ongoing pregnancies of IVP
and MOET, no significant differences
in plasma PSP60 levels were found between
the 4 groups of pregnancies that produced
live, full term calves. Interestingly, plasma
PSP60 levels on day 50 in recipients with
somatic clones that lost their conceptus
between days 50 and 90 were already significantly higher than in recipients with surviving somatic and subsequently nonsurviving embryonic clones. This would
mean that the trophoblast of somatic clones
is more likely to demonstrate aberrant early
cellular differentiation, leading to increased
numbers of binucleate cells. It remains to be
investigated if this is directly associated with
a less developed early vascularization resulting in subsequent placental insufficiency
and foetal death. At later stages (days 150,
180 and 210), the recipients of somatic
clones which had developed a severe
hydroallantois, also had significantly elevated PSP60 levels as compared to nonpathological pregnancies of the control or
cloned groups. Compared with the IVF controls, an increased size of the placentomes
(ultrasonographically measured at 2 week
intervals) was found in the recipients with
somatic clones, but this was not reflected
in altered foetal functions like counted FHR
and growth of the diameter of the foetal
Direct monitoring of foetal functions is
very difficult during the second half of gestation in cattle. Due to the large size of the
foetus, its variable intra-abdominal position
and the limited penetration depth of ultrasound transducers, access to foetal body
structures is rather limited, especially during
the final three months of gestation. This
means that growth curves based on ultrasonographic measurements of foetal structures usually finish around the end of the
second trimester (for details see: Kähn [33];
Ginther [23]). Although the measurements
of the diameter of the foetal eye can be used
to estimate foetal age, there is no evidence as
to whether this parameter accurately reflects
the differences in foetal growth. So, conclusions on the deviations of foetal growth during later stages of development have to be
based on either transversal observations of
collected foetuses or on measurements in
newborns. Under farm conditions, prenatal
identification of abnormal intrauterine development is more likely indicated by the recognition (by external features, transrectal examination and ultrasound observation) of
recipients with hydroallantois, a pathology
which has been reported more often in cows
pregnant from IVP embryos [26, 35]. Additional monitoring of the course of pregnancy
and placental and foetal well-being has to
Monitoring of bovine ET-pregnancies
rely on the measurements of proteins (different PAG’s, PSPB’s) and hormones (progesterone, conjugated and unconjugated
oestrogens, prostaglandin metabolites, cortisol, placental lactogen) in maternal plasma
[34], although their value for the prediction
of abnormal foetal development or foetal
distress needs further exploration.
Prenatal, ultrasound-guided puncture of
foetal fluids has been applied during early
stages of bovine pregnancies [21, 61], but
reports on fluid aspiration during late gestation are not available and might be judged
to be too risky. Under experimental conditions a surgical method (installing catheters
in the foetal umbilical vessels) has been used
to investigate the differences between IVP
and A.I. control foetuses with respect to
blood chemistry and hormone levels during
the final days before delivery [49]. While
IVP foetuses had an elevated haemoglobin
(and as a consequence a higher oxygen content) and lower lactate levels compared to
arterial samples of A.I. foetuses, there were
no differences between the two groups in
arterial oxygen saturation, foetal glucose
tolerance, blood cortisol levels and the
response to ACTH. In fact none of the
observed differences pointed to a poor
preparation of IVP foetuses to extrauterine life, in contrast to the clinical findings reported earlier for several newborn
cloned calves by Garry et al. [22]. Because
both an increased limb length [30] and a
high incidence of flexural deformities of the
limbs [22] have been observed in newborn
calves derived from in vitro produced
embryos, it appears very relevant to explore
foetal motility [23] during IVP pregnancies.
Prenatal restriction of articular mobility is
associated with flexural abnormalities in the
cow [58] and disturbed foetal mobility
around calving could contribute (through
abnormal foetal posture and/or position) to
the higher rates of dystocia reported for IVP
calvings. Also prolonged measurements of
foetal heart rate (FHR), preferably combined with registration of gross foetal
movements, might be useful in this respect,
because different FHR patterns have been
associated with different so-called behavioral states, both in human and animal foetuses (for reviews see: Nijhuis [42]). Significant differences in characteristics of FHR
between acidotic and non-acidotic calves
have been found during calving [31] and
continuous, transcutaneous Doppler measurements of prenatal FHR appear feasible
in the cow [32]. Preliminary data show no
differences in late gestational FHR characteristics between healthy A.I. and IVP foetuses [9], but the value of such measurements for the prediction of prenatal distress
still needs to be assessed in cattle.
While pregnancies obtained from transplantation of in vitro produced embryos suffer from increased (though variable) rates
of embryonic, foetal and perinatal losses
and developmental disturbances, it appears
rather difficult, especially under farm conditions, to identify conceptuses with deviations in development at an early stage.
Transversal studies, collecting conceptuses
at different stages of gestation, have demonstrated that significant differences of foetal
and placentome weight and size may occur
rather early in gestation, but non-invasive
longitudinal ultrasonographic foetometry
and counting of FHR did not reveal significant differences between ongoing pregnancies of MOET and IVP embryos during
the first 4 months. Determinations of maternal plasma levels of placenta-derived pregnancy proteins seem to be more valuable for
a timely detection of abnormal development.
For pregnancy losses between days 24 and
119, a decrease of pregnancy associated protein levels, preceding luteal regression,
occurred slightly more often in IVP than in
MOET recipients of the same herd. This
indicates that pregnancy failures after transfer of IVP embryos are more often the result
of a failure of conceptus development. On
the contrary, abnormally elevated levels of
M.A.M. Taverne et al.
these proteins around day 50 were found to
precede foetal losses in pregnancies from
somatic clones, pointing to a disturbance of
placental cellular differentiation. Because
the uterine contents are more difficult within
reach, direct measurements of foetal and
placental growth during the last trimester
of gestation are restricted. Besides a close
and regular clinical inspection of the dams
and blood sampling for the monitoring of
hormones and pregnancy proteins, transabdominal studies of foetal movements and
FHR might improve the prenatal diagnosis
of aberrant foetal life in cows.
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